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Method for rapidly identifying enpyrene spermatozoon and spermatogenia in silkworm testis

A spermatogonia, silkworm technology, applied in the field of insect biology, can solve the problem of time-consuming and achieve the effect of simple operation

Inactive Publication Date: 2020-04-17
NANYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The document "Dimorphic sperm formation by Sex-lethal" (Hiroki Sakaia, Hiroyuki Oshimab, Kodai Yuric, etc., [J]. PNAS, 2019, 116 (21): 10412-10417) discloses the use of The method of paraformaldehyde fixation and DAPI staining at the same time, but in this method, the testis is dissected in PBS solution to free the sperm, and the fixation and staining time is as long as 1 hour, which is very time-consuming

Method used

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  • Method for rapidly identifying enpyrene spermatozoon and spermatogenia in silkworm testis
  • Method for rapidly identifying enpyrene spermatozoon and spermatogenia in silkworm testis
  • Method for rapidly identifying enpyrene spermatozoon and spermatogenia in silkworm testis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: get five instar silkworm male silkworms, use small surgical scissors and fine-point tweezers to dissect out the testis, and after dissecting out the testis of the silkworm, adopt TC-100 complete medium to remove the tissue (fat body, nerve) of the surface sticky band of the testis of the silkworm The silkworm testis that peeled off is placed in the TC-100 complete culture medium (adding FBS, the final concentration of FBS is 10wt%) of precooling (4 ℃) respectively and tear apart, and the nucleated sperm and spermatogonia are dissociated , placed for 2 hours to obtain a culture solution containing nucleated sperm and spermatogonia;

[0023] Take the above-mentioned culture solution containing nucleated sperm and spermatogonia and spread it evenly on the glass slide, and let it dry.

[0024] Fix with 4% paraformaldehyde in PBS solution for 5 minutes, stain with 10 μg / ml DAPI aqueous solution for 1 minute, cover with a cover slip, mount the slide with glycero...

Embodiment 2

[0029] Example 2 Fixing and staining the silkworm testis to facilitate the identification of nucleated sperm and spermatogonia in the silkworm testis

[0030] The specific steps are as follows: take the fifth instar silkworm male silkworm, use small surgical scissors and fine-tip tweezers to dissect the silkworm testis, place the silkworm testis in 4°C pre-cooled TC-100 complete medium, and remove the sticky tissue on the surface of the silkworm testis (fat body and nerves); take out the testis, put it on a clean glass slide, tear the testis of the silkworm with tweezers, apply the contents of the testis of the silkworm evenly on the slide, and let it dry in the air.

[0031] Fix with 4% paraformaldehyde in PBS for 5 minutes, stain with 10 μg / ml DAPI aqueous solution for 1 minute, cover with a cover glass, mount the slide with glycerol, and observe with an upright fluorescent microscope. Fluorescence microscope observations showed that figure 2 shown.

Embodiment 3

[0032] Embodiment 3 paraformaldehyde fixed time screening

[0033] Take the fifth instar silkworm male silkworm and use small surgical scissors and fine-tip tweezers to dissect out the silkworm testis, place the silkworm testis in 4°C precooled TC-100 complete medium (with FBS added, the final concentration of FBS is 10wt%);

[0034] Remove the fat body and nerves attached to the surface of the silkworm testis, take out the silkworm testis and place it on a clean glass slide, tear the silkworm testis with tweezers, apply the contents of the silkworm testis evenly on the glass slide, and let it dry in the air.

[0035] Fix with 4% paraformaldehyde in PBS solution for 5min, 10min, 15min, 30min and 1h respectively, stain with 10μg / ml DAPI for 10min, cover with a cover glass, mount the slide with glycerol, and detect the different fixation of paraformaldehyde with an upright fluorescence microscope Effect of time on staining of nucleated spermatozoa and spermatogonia in silkworm. ...

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Abstract

The invention belongs to the field of insect biology, and particularly relates to a method for rapidly identifying enpyrene spermatozoon and spermatogenia in a silkworm testis. The method comprises the following steps of: putting the silkworm testis obtained by dissecting into a TC-100 complete culture medium to remove tissues adhered to the surface of the testis, tearing off the silkworm testis,smearing, fixing and dyeing a sample containing the enpyrene spermatozoon and spermatogenia, and observing the sample under a fluorescence microscope. According to the method, the complete contours ofthe enpyrene spermatozoon and spermatogenia can be effectively maintained in the operation process, and identification of the enpyrene spermatozoon and spermatogenia can be achieved through photographing observation under fluorescence microscopy.

Description

technical field [0001] The invention belongs to the field of insect biology, and in particular relates to a method for rapidly identifying nucleated sperm and spermatogonia in the testis of the silkworm. Background technique [0002] Sperm has always been a research hotspot for physiologists, morphologists and cell biologists. A typical sperm cell consists of a head (nuclear acrosome), neck (mitochondrial sheath) and tail (centrosome). Sperm played an important role in the development of evolutionary theory. [0003] Bombyx mori, like other Lepidoptera insects, has sperm dimorphism, that is, nucleated sperm and anucleated sperm can develop from spermatogonia. Nucleated sperm can fertilize eggs and develop into individuals, while non-nucleated sperm cannot fertilize eggs and play an auxiliary role in the fertilization process of nucleated sperm. Both nucleated and anucleated sperm are developed from spermatogonia of the same genotype, but they are quite different in shape,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N1/30
CPCG01N21/6458G01N1/30G01N2001/305
Inventor 李丹丹王艳艳常美玲程浩阚云超王艺付裕霍春月马田田
Owner NANYANG NORMAL UNIV
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